The determination of the amino acid sequence of proteins is very important for modern molecular biology. Proteins and peptides are essential components of all living cells. They are the structural elements of cell walls and cell membranes, enzymes, immunoglobulins/antibodies, transport molecules and most hormones. The building blocks of proteins are the 20 natural amino acids that are covalently linked together via amide bonds and thus form linear protein chains. The amino acid sequence (primary structure) determines the very complex secondary and tertiary structures responsible for the biological functions of the proteins.
The sequence of a protein or peptide can, in principle, be deciphered by a stepwise chemical or enzymatic degradation from either the amino- (N-) or carboxyl- (C-) terminal end. Single amino acids are removed one by one from the polypeptide chain, separated and identified. Although several methods for a stepwise chemical or enzymatic degradation have been elaborated (see: Elizinga, M. editor, Methods in Protein Sequencing, Humana Press, Clifton, N.J., 1982) the preferred method was introduced by Edman in (Acta Chem. Scand., 4:283 (1950)).
In the method described by Edman, the chemical procedure for removing one amino acid residue from a polypeptide chain consists of three steps. First the polypeptide is reacted at its amino-terminus with an isothiocyanate (ITC), e.g. phenylisothiocyanate (PITC), in a solvent under basic or anhydrous conditions to form a phenylthiocarbamate (PTC). This step is generally referred to as "coupling". Second, under the influence of anhydrous acid (e.g., trifluoroacetic acid, TFA), the PTC cyclizes to an anilinothiazolinone (ATZ) with concomitant cleavage of the last N-terminal amino acid from the polypeptide chain. This step is generally referred to as "cleavage". This provides exposure of the amino groups of the next, adjacent amino acid in the polypeptide chain for the following reaction cycle. The ATZ derivatives are relatively unstable and therefore, in the third step, are converted to the stable phenylthiohydantoins (PTH) by treatment with aqueous acid (e.g., TFA). This step is generally referred to as "conversion". The chemistry of one cycle is shown in the reactions below. ##STR1## Conditions have been determined for separating the 20 PTH derivatives of the natural amino acids of high performance liquid chromatography (HPLC). Due to the specific retention times of the 20 PTH amino acid derivatives, the amino acid being removed by each respective cycle can be identified.
The polypeptide, which is shortened by one amino acid, is subjected to the next degradation cycle to furnish the second amino acid derivative in the sequence. The sequence is determined by repeating the foregoing steps for each of the amino acids in the polypeptide chain.
Several variations of the Edman chemistry have been proposed: The `liquid phase method` described above has been automated as a `spinning cup automatic sequencer` (Edman, P. and Begg, G., Eur. J. Biochem., 1:80 (1967) and U.S. Pat. No. 3,725,010).
The first use of solid supports in Edman peptide sequencing was described by Schroeder in Methods in Enzymology, 11:455 (1967). In this method, the peptide or protein was applied to a paper strip. The material remained adsorbed to the paper, without the formation of covalent bonds, throughout the sequencing process. Coupling and cleavage were performed by exposing the paper strip and adsorbed polypeptide to gas phase reagents. As such, this process is referred to as gas phase sequencing. The process has been automated as described by Hewick, R.M. et al. in J. Biol. Chem., 256:7990 (1981). Unfortunately, this method worked satisfactorily only for proteins which remained insoluble on the surface of the paper strip during the sequencing reactions. The method therefore had to be performed under very controlled conditions.
As a result of the above limitations, the solvents used in the sequencing must be volatile and carefully selected to allow quantitative removal of reagents after the coupling step. To maximize the signal, cleavage and transfer of the ATZ derivatives must be performed without elution of any of the protein from the solid support. Additionally, all solvents and reagents used must be very pure, since they could only be removed from the paper surface by drying/evaporation. Any remaining non-volatile impurities might interfere with subsequent determination of PTH derivatives.
Unfortunately, cycle time for the sequencing is often long, since reactions in the gas phase are slow. This may be because contact of reagents with the protein is mediated only by convection. Due to the fact that the process employs gas-phase reagents, agitation and/or the use of liquid solvents that would promote fast reaction rates, complete extraction of reagents, and removal of ATZ derivatives is not possible. In addition, the solubility of the 20 ATZ and PTH derivatives differs significantly due to the different properties of the 20 distinct amino acid side chains. The efficient removal of the derivatives from the solid support without loss of non-covalently bound protein is therefore difficult.
Thus, sequence data can only be obtained by compromising reaction conditions. This leads to long reaction times and also results in inefficient reactions and solvent extractions. Consequently, only relatively short peptides can be sequenced by this method. Furthermore, the gas phase sequencing method is not suitable for high sensitivity sequencing of small amounts of protein (in the picomol range).
Although the gas phase sequencing method has been significantly improved by the adsorption of proteins to Polybrene-coated (Polybrene is a trademark of Abbott Laboratories) glass fiber filters (Hewick, R.M. et al., J. Biol. Chem., 256:7990 (1981) and Tarr, G.E. et al., Anal. Biochem., 84:622 (1978)), there are still serious drawbacks. For example, the Polybrene-coated glass fiber filter has to be extracted by performing 3 to 5 degradation cycles before sample application. This precycling reduces the background of some impurities to a practical level, but several UV-absorbing contaminants remain which interfere with coeluting PTH derivatives during HPLC separation and identification. This results in unpredictable and relatively low initial yields: for sperm whale myoglobin, for example, initial yields of 25% (Esch, F.S., Anal. Biochem., 136:39 (1984)) and 78% (Hawke, D.H. et al., Anal. Biochem., 147:315 (1985)) have been reported. In addition to this, the other previously mentioned disadvantages still apply. These disadvantages result from compromises to prevent a wash-out of the non-covalently trapped polypeptide. Due to the numerous dry-down steps to remove solvents and reagents, impurities are concentrated onto the surface, necessitating the use of ultrapure reagents and solvents.
The covalent attachment of 3-aminopropyl or N,N,N-trimethylammoniumpropyl groups to glass fiber filters has improved the situation to some extent, in that precycling is not necessary and initial yields of PTH amino acid derivatives are higher (Aebersold, R.H. et al, J. Biol. Chem., 261:4229 (1986)). The background level has also been significantly lowered due to the strong electrostatic immobilization of the proteins on the glass fiber surface.
In a recent patent (U.S. Pat. No. 4,665,037) a liquid-solid affinity chromatography which solves some of the aforementioned problems is described. However, no protein sequencing results are given to demonstrate the utility of the method. Additionally, the various affinity supports and reagents are difficult to synthesize and automation of this process appears to be difficult. Furthermore, in the process described, the peptides and proteins have to be chemically modified prior to the sequencing process to block some side chain functionalities which otherwise would interfere with the affinity reagents.
Most of the disadvantages of the previous methods and materials result from the non-covalent nature of the immobilization of the polypeptides onto the solid support. Covalent attachment of proteins and peptides to the solid support should allow for a superior sequencing procedure to be employed. This procedure is referred to as solid phase sequencing.
Two sets of problems have to be addressed for solid phase sequencing to be practical:
(1) the choice of polymer support and PA0 (2) the method of covalent immobilization to the support. PA0 (1) The protein or peptide is dissolved in 0.2-0.8 ml 50 mM sodium bicarbonate buffer pH 8.0 (perhaps containing 1-2% w/v SDS to aid solubility) and the solution added to 10-100 mg DITC APG (11). PA0 (2) Following incubation for 2-4 hr at 40-50.degree. C., 0.2 ml n-propylamine is added to block excess isothiocyanate groups on the support. PA0 (3) The glass/protein suspension is then centrifuged and the supernatant (containing any unbound peptide) is recovered. PA0 (4) The glass beads are then transferred to a small fritted filter funnel and washed with bicarbonate and methanol (containing traces of triethylamine, TEA). PA0 (5) The washed support is then dried by applying light vacuum for a few minutes, and packed dry into the sequencer reaction column.
Various supports have been used in conjunction with Edman protein sequencing chemistry.
For example, several porous solid supports have been based upon a polystyrene backbone having functionalized phenyl groups. Representative reactions for the conversion of the phenyl group containing materials to useful solid phase sequencing supports are given below: ##STR2##
In the first reaction above, polystyrene having phenyl groups has been functionalized with chloromethyl groups to form material (1). This is then transformed by treatment with ethylenediamine to (2-aminoethyl)-aminomethyl polystyrene designated resin (2). (Laursen, R.A., Eur. J. Biochem., 20:89 (1971). Although peptides could be attached in high yields to this support, sequence overlaps were significant due to incomplete cleavage in the TFA step because of poor penetration of the acid into the hydrophobic polymer. Addition of an amino group to the backbone phenyl ring to give resin (3) resulted in greatly improved swelling in TFA (Laursen, R.A. et al, FEBS Letters, 21:67 (1972)). Under the acidic conditions provided by TFA the amino groups are protonated and thus the polarity of the resin is increased.
Further development of the diisothiocyanate attachment method is described by Laursen et al. in FEBS Lett., 21:67 (1972). Aminopolystyrene resin (4) was demonstrated to be equally satisfactory as resin (3) for peptide attachment. Preparation of (4) was much easier than (3), so it rapidly became the resin of choice for this method of attachment. However, aminopolystyrene resin proved to be less efficient for the immobilization of peptides by certain carboxyl activation chemistries due to the relatively poor nucleophilicity of aryl amines.
Horn and Laursen in FEBS Letters, 36:285 (1973)) developed the triethylenetetramine (TETA) resin (5) which showed many of the desirable properties of (3) with the added attraction of much simpler synthesis. The long chain aliphatic amino groups were also much more accessible to reagents as well as peptides and proteins Some stability problems, however, were noted after a few months of storage, even under refrigeration.
Although used successfully in much of the early work in solid-phase protein sequencing, the polystyrene-based resins exhibit several inherent properties which increase the difficulty of both preparation and use. For example, the degree of both substitution and crosslinking must be carefully controlled. Most of the successful resins were derived from resins crosslinked with 1-2% divinylbenzene (Merrifield resin). Greater degrees of crosslinking resulted in resins with drastically reduced reagent permeability. Chloromethyl substitutions of around 15% were found most practicable; increasing this level simply resulted in increased crosslinking of methylene bridges (Patterson, J.A., in `Biochemical Aspects of Reactions on Solid Supports`, Stark, G.R., editor, Academic Press, New York, 1971, pp. 189).
Solvent-induced swelling of the resins in certain solvents (such as pyridine or TFA) followed by shrinkage in others (e.g., methanol) during a typical Edman degradation cycle resulted in considerable problems due to blockage and solvent channeling when the resin was packed directly in reaction columns. This problem was partly resolved by Laursen (see Laursen, Eur. J. Biochem., 20:89 (1971)) by mixing the resin with a fifty-fold excess of glass beads prior to column packing. This allowed for swelling to occur harmlessly in the interstices between the beads. The issue was addressed more directly by Inman et al. (in `Solid Phase Methods in Protein Sequence Analysis`, Previero, A. and Coletti-Previero, M.A., editors, North Holland Press, Amsterdam, 1978, pp 81) who prepared resins derived from highly crosslinked polystyrene. The resulting material was largely incompressible, was very resistant to solvent-induced volume changes and possessed a large reagent-accessible surface area.
One further deficiency of the earlier polystyrene resins was that the matrix did not permit penetration of large proteins or peptides. The practical limit for efficient attachment was generally held to the peptides of only 30-50 residues in length (Laursen, R.A. and Machleidt, W., `Methods of Biochemical Analysis`, Glic,, D., editor, 26, pp 201 (1980).
In addition to the polystyrene resins, polyacrylamide supports have also been used for solid-phase sequencing. Representative reactions with polyacrylamides are given below: ##STR3## Early experiments resulted in a hydrophilic sequencing resin (6) prepared from polyacrylamide beads (Bio-Gel P, Bio-Rad Laboratories) and ethylenediamine. Cavadore et al., FEBS Letters, 66:155 (1976), prepared and used a resin similar to (6) from Bio-Rex 70 (a weak cation exchanger) by conversion to the acid chloride followed by reaction with ethylenediamine. Cavadore and Vallet, Anal. Biochem., 84:402 (1978), prepared another hydrophilic activated polyacrylamide (8) by coupling 1,4-diaminobenzene sulphonic acid to polyacrylyl chloride. Subsequently, the free arylamino groups were converted to isothiocyanate in situ. The sulphonic acid residues imparted hydrophilic character to the resin and increased non-covalent protein binding by ion-exchange.
Atherton et al., FEBS Letters, 64:173 (1976) also prepared a polyacrylamide support with a beta-alanylhexamethylendiamine side chain (7) which was used as a sequencing support when mixed with glass beads to overcome flow problems.
Although capable of binding larger peptides and proteins than the earlier polystyrene supports, the polyacrylamide-based resins still suffered from significant flow problems. These resulted from solvent-induced swelling and shrinking of the supports which caused blockage of the fluid flow pathways. The resins were sensitive to prolonged, repeated exposure to TFA which resulted in increased levels of contamination eluting from the columns after several sequencing cycles. After some initial interest, none of the above polyacrylamide resins have been used extensively in sequencing (Laursen, R.A. and Machleidt, W., `Methods of Biochemical Analysis`, Glick, D., ed., 26, pp. 201 (1980)).
Largely because of the poor properties and limited accessible surface area of the polymeric supports mentioned above, by far the most successful supports for solid-phase sequencing have been prepared by the derivatization of controlled pore glass (CPG) with alkylsilanes (see Machleidt, W. and Wachter, E., Methods in Enzymol., 47:263 (1977)). A variety of supports based on these materials are shown below: ##STR4## Two types of CPG supports have been used extensively: 3-aminopropyl glass (APG), designated (9), prepared by reaction of CPG with 3-aminopropyl triethoxysilane (Robinson, P.J. et al., Biochim. Biophys. Acta, 242:659 (1971); Wachter, E. et al., FEBS Letters, 35:97 (1973)) and N-(2-aminoethyl)-3-aminopropyl glass (AEAPG), (10), obtained by reaction of CPG with the corresponding trimethoxy silane (Bridgen, J., FEBS Letters, 50:159 (1975)). Controlled pore glass with a nominal pore size of 75 .ANG. was held to be suitable for most solid-phase sequencing applications (Laursen, R.A. and Machleidt, W. (1980), see above) although more efficient attachment of larger proteins (greater than about 30,000 daltons) or proteins in the presence of detergents could be achieved using CPG supports of up to 500 .ANG. nominal pore size (e.g., Bridgen, J., Methods in Enzymol., 47:321 (1977)).
The principal superiority of solid-phase protein sequencing comes mainly from the greater flexibility in the choice of conditions for the sequencing chemistries. However, solid-phase protein sequencing in the past was severely restricted because efficient methods to immobilize peptides and proteins on the solid support were not available, particularly when working with very small amounts.
A number of methods have been employed: Horn and Laursen, FEBS Letters, 36:285 (1973), immobilized homoserine lactone terminated fragments obtained after cyanogen bromide cleavage of proteins to amino supports. L'Italien and Strickler, Anal. Biochem , 127:198 (1982) have used water-soluble carbodiimides to selectively covalently attach peptides through their alpha-carboxyl groups to amino supports. The attachment of peptides through their tyrosine side chains using diazophenyl supports has been investigated by Chang et al., FEBS Letters, 84:187 (1977). The two amino-functional supports APG (9) and AEAPG (10) have also served as precursors to link peptides or proteins to the solid support via the sulfhydryl groups of cysteine residues. This was accomplished by reacting APG with iodoacetic acid in the presence of dicyclohexylcarbodiimide (DCCI) to produce the iodo-glass support (13). Little or no side reaction was observed with the hydroxyl groups of serine, threonine and tyrosine or with methionine (Chang, J.Y. et al., FEBS Letters, 78:147 (1977)). An aryl amino support (14) was prepared by reaction of APG (9) with p-nitrobenzoyl chloride followed by reduction to the aryl amine with sodium dithionite (Weetall, H.H., Biochim. Biophys. Acta, 212:1 (1970). This support has proven to be particularly suitable for attachment of peptides following activation of carboxyl functions with carbodiimides. A review describes the various methods of attachment in more detail (Laursen and Machleidt `Methods of Biochemical Analysis`, Glick, D., ed., 26, pp. 201 (1980)).
By far the most widely used method to attach peptides and proteins to solid supports involves the epsilon amino group of lysine residues with activated isothiocyanate supports such as (11) or (12). These supports are prepared by reaction of either APG (9) or AEAPG (10) with p-phenylene-1,4-diisothiocyanate (DITC) (Wachter, E. et al. (1973)). Reaction of the amino groups of proteins with the isothiocyanate moiety is readily achieved in mildly basic aqueous solution (pH 8) at temperatures from 25-55.degree. C. The presence of detergents (e.g., sodium dodecyl sulphate (SDS)), or chaotropic agents such as 8M urea or saturated guanidinium hydrochloride have no significant effect on the attachment chemistry (Machleidt, W. et al., `Advanced Methods in Protein Sequence Analysis`, Wittmann-Liebold, B., Salnikow, J. and Erdmann, V.A., editors, Springer-Verlag, Berlin, Heidelberg, pp. 91 (1986). Consequently, attachment of proteins or peptides to DITC supports is generally accepted as the method of choice for solid-phase sequence analysis.
The advantages of CPG supports over the earlier polymeric resins are numerous. The very large surface area per unit weight (typically 100 square meter/g for 200-400 mesh CPG of 200 .ANG. nominal average pore-size) greatly improves the attachment capacities for proteins and peptides (capacities of 0.2 mmol amine/g support are typical; Schmitt, H.W. and Walker, J.E., FEBS Letters, 81:403 (1977)). The functional groups on the porous glass supports are accessible to solvents of all kinds and the rigid inorganic matrix is incompressible for all practical purposes Supports prepared from 200-400 mesh CPG beads provide an efficient packing of continuous-flow reaction columns, with very low fluid back-pressure.
The coupling medium can be freely selected according to the solubility of peptides and proteins and the requirements of the coupling chemistries: for example, the hydrophobic DCCD-binding subunit of mitochondrial ATP-ase was attached to DITC activated APG (11) in a mixture of chloroform and methanol (Wachter, E. and Weerhahn, R., `Solid-Phase Methods in Protein Sequence Analysis`, Previero, A. and Coletti-Previero, M.A., editors, North Holland Press, Amsterdam, pp. 185 (1978)).
The porous glass beads can be used in reaction columns with aqueous as well as anhydrous organic solvents. The physical stability of the glass beads has facilitated the design of efficient Edman degradation chemistries (Walker, J.E., et al., Biochem. J., 237:73 (1986)) with significant improvements in speed and repetitive stepwise yields.
Apart from slight problems with slow hydrolytic loss of surface siloxane groups at the elevated temperatures (up to 56.C) and basic conditions (pH 10-11) required for part of the typical Edman degradation (perhaps losing up to 0.5% of attached peptide per cycle), the principle disadvantages to the use of glass beads for solid-phase sequence analysis are related to the practical problems of sample preparation (coupling of the peptide or protein) and handling.
A typical series of steps involved in sample preparation is as follows:
Similar techniques are used for the polystyrene-based supports described previously. In each case, these procedures require careful attention and manipulative skills, particularly when working with small quantities of beads (typically less than 10 mg).
The use of beaded supports (either CPG or polystyrene resin) also precludes the use of electroblotting techniques for the recovery of proteins from SDS polyacrylamide gels, which require the transfer onto a sheet or membrane support.
While the above materials and methods for sequencing peptides and proteins have been quite valuable, many of them are unable to meet the current requirements in molecular biology. Recent developments in this field demand the determination of protein and peptide sequences in the picomol range and below. This is because many proteins of great biological interest can only be obtained in these small quantities. Establishing a short peptide sequence at the N-terminus of a protein enables one to initiate experiments aimed at identifying the corresponding gene and generating the entire protein sequence by determining the DNA sequence. Complex protein patterns from normal cells or cells derived from various diseased tissues (such as cancer, degenerative nervous diseases (Harrington, M.G. and Merrill, C.R., Clinical Chemistry, 30:1933 (1984)) and genetic diseases) can be visualized by isolating proteins in 1-10 nanogram quantities using 2D polyacrylamide gel electrophoresis (O'Farell, P.H., J. biol. Chem., 250:4007 (1975). To obtain sequence information from such rare proteins does not allow the type of sample manipulation described above for the beaded supports. The availability of a flat support onto which the respective proteins can be directly blotted after ID and 2D polyacrylamide gel electrophoresis is a necessity. Additionally, efficient methods to covalently immobilize these very small amounts of proteins onto the flat support during the blotting process are needed as well. Thus isolation of proteins by electroelution from polyacrylamide gel pieces or by HPLC methods, (which are laborious and very often result in very low recovery yields), can be avoided.
Aebersold et al., J. Biol. Chem., 261:4229 (1986); Kent, S. et al., BioTechniques, 5:314 (1987); Vandekerckhove, J. et al, Eur. J. Biochem., 152:9 (1985) derivatized the surface of glass-fiber sheets with 3-aminopropyl trimethoxy silane and from this prepared the activated isothiocyanate surface by reaction with a DITC analog to form glass-fiber (11). The DITC glass-fiber sheets were successfully used to covalently attach and sequence proteins electroblotted directly from SDS polyacrylamide gels (Aebersold et al., (1986), see above). Significant drawbacks of glass-fiber papers are the difficulties of detecting proteins using the popular Coomassie Blue for staining (Aebersold et al. (1986), see above), the non-optimal texture which does not allow for an intimate contact and undistorted transfer of material during the blotting process, the very low capacity to bind proteins, (which is in the range of 7-10 ug per cm.sup.2), and the fact that both the repetitive yield and initial yield for coupling of proteins electroblotted onto glass-fiber sheets decreases with the decreasing amount of proteins present (Yuen, S. et al., Applied Biosystems User Bulletin, 24 Foster City, CA (1986)).
As the coupling reaction between the peptides or proteins and the ITC group on the surface of the glass fiber paper is slow, a trapping process for the peptides or proteins would increase the efficiency of coupling; DITC-glass fiber paper can provide this only by electrostatic interaction with the amino groups which were left over following the reaction with DITC. As there could be also a repulsion operating between the ammonium groups on the glass fiber surface and similarly charged groups on the surface of the peptide or protein this is an unpredictable, variable and inefficient process. It is obvious that DITC-glass fiber sheets are particularly inefficient in coupling small peptides and apparently are not suitable for protein sequencing in the picomol range and below.
Other flat materials which provide for a more efficient electroblotting of proteins are nitrocellulose (Towbin, H. et al., Proc. Natl. Acad. Sci. USA, 76:4350 (1979)), diazophenyl paper (Renart, J. et al., Proc. Natl. Acad. Sci. USA, 76:3116 (1979) and charge-modified nylon sheets (Gershoni, J.M. and Palade, G.E., Anal. Biochem., 124:396 (1982). Nitrocellulose has not been functionalized to allow for covalent attachment of proteins and, in addition, dissolves during a variety of sequencing reactions. When subjected to the Edman degradation chemistry, the charge-modified nylon collapses into a solid pellet, while diazophenyl paper is physically and chemically not stable enough to withstand the repeated cycling associated with the sequencing method. Diazophenyl paper also limits the attachment of a protein to cases in which relatively rare tyrosine residues are present.
Recently, polyvinylidene difluoride (PVDF; also known in the art as polyvinylidene fluoride (PVF)) membranes have been used for efficient electroblotting and gas-phase sequencing (Matsudaira, P., J. Biol. Chem., 262:10035 (1987); Pluskal, M.G. et al., BioTechniques, 4:272 (1986)). Compared to glass fiber sheets derivatized with ammonium groups, the capacity of PVDF membranes to bind proteins is significantly better (170 ug/cm.sup.2 for PVDF versus 7-25 ug/cm.sup.2 for derivatized glass fiber sheets; see Matsudaira, 1987). The initial sequencing yield for proteins adsorbed to PVDF (76-97%) also compared very favorably with the 15-25% initial yields obtained for proteins electroblotted onto glass fiber sheets (Matsudaira, 1987, see above).
The very efficient and high adsorption properties of PVDF membranes for proteins has several other advantages. Protein solutions can be rapidly desalted by dot-blotting onto the membrane surface and washing with water to remove buffer salts. In addition to this, dilute protein samples can be concentrated several fold on the membrane surface through multiple loadings.
PVDF has been used for direct gas phase protein sequencing (Matsudaira, J. Biol. Chem., 262:10035 (1987). The mechanism of protein adsorption to PVDF is not known, but may result from hydrophobic or dipole interaction between proteins and the polymer surface. Thus, the disadvantages described above, resulting from non-covalent attachment of peptides and proteins to a solid support, apply to PVDF as well.
U.S. Pat. No. 4,340,482 describes a process for grafting amino acid molecules under highly basic conditions onto the surface of the preformed PVDF membrane. Apparently under strong basic conditions (particularly in the presence of a phase transfer catalyst) hydrogen fluoride is eliminated and carbon double bonds are formed exclusively on the surface of the PVDF membrane (Dias, A.J. et al, Macromolecules, 17:2529 (1984). The reaction of amines with fluorocarbon polymers was also described by Bro in J. Appl. Polymer Sci., 1:310 (1959)). Bro suggests that under the influence of the amine, hydrogen fluoride (HF) is eliminated followed by an addition of the amine to the double bond. Several reactions may follow including cross-linking and the elimination of another HF molecule at the addition site of the first amine to form an imino structure which can tautomerize to an enamine structure (Smith, J.R. et al., J. Appl. Polymer Sci., 5:460 (1961)). Aqueous KOH is apparently significantly less reactive to eliminate HF than amines (Chambers, R.D. et al., Tetrahedron Letters, 10:629 (1963).
The process described in U.S. Pat. No. 4,340,482, by which amino acids are apparently linked covalently via their amino functions to the surface of PVDF renders the hydrophobic PVDF surface hydrophilic. A different way to modify the properties of a polymer surface is described in U.S. Pat. 4,618,533. In this patent, a permanent coating is grafted and/or deposited onto the surface of a membrane by the copolymerization of appropriate monomers onto all the solvent-accessible surface area of the microporous structure. If monomers such as hydroxypropylacrylate are used as the coating, a hydrophobic surface can be transformed into a hydrophilic surface.
In co-pending U.S. Pat. application Ser. No. 093,011, filed Step. 4, 1987 now U.S. Pat. No. 4,923,901 (Koester, H. and Coull, J.H., entitled `Membranes with Bound Oligonucletides and Peptides`) chemically derivatized membranes linked to protected nucleosides or amino acids are described. These membranes are useful for the synthesis of nucleic acids and peptides.